P < 0.05). There were no differences between the groups in terms of postoperative IOP and visual outcomes or in astigmatism preoperatively or postoperatively . In both groups, the mean preoperative and postoperative astigmatism were ATR and OBL, respectively. Preoperative and postoperative centroids in the P-Ex-PRESS group were 0.44 ± 1.32 D at 177° and 0.35 ± 1 D at 8°, respectively, (P = 0.5) and in the P-Trab group were 0.16 ± 1.5 D at 141° and 0.39 ± 1.38 D at 29°, respectively (P = 0.38). Both P-Ex-PRESS and P-Trab showed comparable antihypertensive efficacy in treating open-angle glaucoma over 6 months. Preoperative and postoperative astigmatism did not differ between groups. The groups showed comparable results for final visual acuity.The aim of this study was to compare surgical and refractive outcomes between phacotrabeculectomy (P-Trab) and phaco with Ex-PRESS (P-Ex-PRESS) for glaucoma at a 6-month follow-up. This prospective randomized controlled trial included 81 eyes; 43 eyes (53%) and 38 eyes (47%) were assigned to the P-Ex-PRESS and P-Trab groups, respectively. Refraction, intraocular pressure (IOP), and best-corrected visual acuity were measured. Refractive change was analyzed using the cylinder’s magnitude, and polar analysis assessed the change in the trend of astigmatism , evaluating mean astigmatism in centroid form. All patients showed a statistically significant postoperative decrease in IOP ( Trabeculectomy and implantation of the Ex-PRESS mini device  are anti-glaucoma procedures that improve subconjunctival outflow . AlthougThis study adheres to the tenets of the Declaration of Helsinki and the Principles of Good Clinical Practice developed by the European Union. The study protocol was approved by the Bioethics Committee at the Medical University in Białystok under the number R-I-002/443/2014 and registered on clinicaltrials.gov (registration number: NCT04335825). Written informed consent to participate for at least 6 months was obtained from all patients after an explanation of the nature of the procedure and surgical alternatives. The study protocol was similar to that documented in our previous work .We recruited consecutive patients who were referred to the ophthalmology clinic of our hospital and qualified for combined surgery. Glaucoma with coexisting cataract graded NC1 or NC2 by the Lens Opacities Classification System III scale was the surgical indication. Patients with primary open-angle glaucoma, pseudoexfoliation glaucoma, and pigmentary glaucoma, where target IOP was not achieved, despite maximally-tolerated topical and systemic medication and well-documented visual field defect progression, were eligible for treatment. A patient qualified for surgery if any of the following additional inclusion criteria was present: significant diurnal variations in IOP, poor patient compliance, or allergy to topical anti-glaucoma drugs with progressive visual field loss. The exclusion criteria were as follows: lack of consent for study participation, history of eye surgery or laser procedures within the eye, closed or narrow-angle glaucoma, diabetes, advanced macular degeneration, and active inflammatory disease.The randomized prospective study included 81 eyes of 81 patients. A computer-generated randomization list was created, with allocation concealment. Randomization was performed using sealed envelopes, opened on the day of surgery, to determine the randomization group. The eyes were individually randomized in a 1:1 ratio to either phacoemulsification with simultaneous Ex-PRESS mini glaucoma shunt implantation (43 eyes (53%)) or P-Trab (38 eyes (47%)).Detailed data on patient demographics , previous treatments, and surgical procedures were collected at the time of qualification. Before surgical treatment, all patients underwent a basic examination, which included determination of IOP, uncorrected distance visual acuity, best-corrected visual acuity (BCVA), and refractive findings (refractive error: sphere and cylinder with axis), axial length, and biomicroscopic examination of the anterior and posterior eye segments, with a detailed assessment of the retina and optic nerve disc. The BCVA was examined with the Snellen notification and expressed as logMAR units. A Snellen BCVA of 1.0 (100% or 20/20) equals a logMAR of 0.The intraocular lens (IOL) power was calculated with the IOL Master 700 . All patients were implanted with the same type of IOL. Gonioscopy was performed along with the field of vision test. The IOP was measured during preoperative examination using a slit-lamp–mounted Goldmann applanation tonometer in accordance with the Advanced Glaucoma Intervention Study. The reading in mm Hg was rounded to the closest integer. Each measurement was repeated twice, and if the difference between the two readings was ≥3 mm Hg, a third measurement was taken. The mean of two or three measurements was used to determine the IOP.All surgical procedures were performed under retrobulbar anesthesia (2% xylocaine and 0.5% bupivacaine) by the same experienced surgeon (JK) for the duration of the study. In both procedures, the fornix-base conjunctiva was dissected, and the sclera was exposed. A limbus-based, square-shaped (4 mm × 4 mm) scleral flap was dissected using the technique described by Traverso et al. . A clearDuring the follow-up visits, the IOP and BCVA were measured, as mentioned above, by the same unmasked resident doctor. Detailed biomicroscopic evaluation of the anterior chamber and fundus of the eye was performed. During the postoperative assessment, both complications and the number of IOP-lowering medications administered were documented. Additional procedures were performed when insufficient filtration was noted as an elevated IOP (≥16 mm Hg) or an underdeveloped or completely flat filtering bleb . InadequThe success rate was defined as either complete or qualified. Complete surgical success was defined as IOP ≤18 mm Hg without anti-glaucoma medications, whereas qualified success was defined as IOP ≤18 mm Hg with a maximum of two anti-glaucoma medications (determined by the number of active ingredients). In the case of IOP >18 mm Hg with or without anti-glaucoma medications or when the eye required further surgical intervention, surgery was considered a failure. No anti-glaucoma medications were allowed on the day of the operation. When surgery did not achieve the expected results, medications were re-administered as recommended by the European Glaucoma Society rules. Control examinations were performed both before treatment and on the first and seventh postoperative days, as well as at postoperative days 30, 90, and 180. Postoperatively, all eyes were treated with topical postoperative steroids , and tapered to twice daily after 1 week. Antibiotic (Moxifloksacin) was administered as a single drop three times daily for 2 weeks and nonsteroidal anti-inflammatory drugs as one drop three times daily for 4 weeks.Autorefractometry data from before and 180 days after surgery were included in the analysis. Refractive and vector analyses were performed. Refractive analysis, a simple arithmetic calculation of the mean of the cylinder without considering its axis, was performed to compare with the numerical results available in the literature. This difference in the mean value of the cylinder allows for reporting the mean change in the magnitude of astigmatism. All calculations were performed using the plus form of the cylinder.The vector analysis is a calculation of the cylinder change considering its axis. The preoperative and postoperative refractive measurements (cylinder with its axis) were evaluated by vector analysis, according to the method proposed by Holladay et al. . Data weFor conversion from polar to Cartesian values, the following mathematical formulae were applied:2 + y2)cyl = √(x−1 (y/x)]angle = ½ × [tanif x > 0 and y > 0→axis = angleif x < 0→axis = angle + 90°if x > 0 and y < 0→axis = angle + 180°if x = 0 and y < 0→axis = 135°if x = 0 and y > 0→axis = 45°if x = 0 and y = 0→axis = 0°if y = 0 and x < 0→axis = 90°if y = 0 and x > 0→axis = 0°.Cartesian coordinates were converted to standard polar values using the below formulae:This calculation was performed for each individual to obtain the surgically induced refractive change. The mean of all x and y values were calculated to allow calculation of the aggregate refractive change of the analyzed groups. Data were displayed as double-angle plots because the angles were doubled owing to the return of the astigmatism vector to the same value when traversing 180°.The major and minor axes of the centroid of the ellipse were determined by calculating the standard deviations of the x and y coordinates. The astigmatism trend was evaluated based on the shape factor and the centroid axis.The aggregate data considered the mean value of astigmatism (mean magnitude of the cylinder), which does not include the axis analysis or centroid calculation.The centroid was used to define the direction of astigmatism .The double-angle plots were prepared using the double-angle-plot-tool for astigmatism available on the American Society of Cataract and Refractive Surgery website. Data on astigmatism values were depicted in cumulative data plots at 0.5-D intervals.t-test or Mann-Whitney U test. The x2 test of independence for two variables was used to compare quantitative characteristics. Values of P < 0.05 were considered to indicate statistical significance. Analyses were conducted using SPSS version 24.0 .In both groups, quantitative data are expressed as arithmetic means, standard deviations, and medians. Qualitative characteristics are expressed as numbers and percentages. Data were tested for normality using the Shapiro–Wilk test. Between-group comparisons were made using Student’s A total of 43 and 38 patients underwent P-Ex-PRESS and P-Trab procedures, respectively. The demographic data are summarized in The mean IOP levels before and after surgery are summarized in n = 8) and 22.6% (n = 8) of patients, respectively. However, during the follow-up, 50% (n = 21) and 75% (n = 28) of patients were drug-free in the P-Ex-PRESS and P-Trab groups, respectively.The average numbers of IOP-lowering medications before and after surgery are summarized in n = 19) and 49% (n = 18) of patients achieved complete success in the P-Ex-PRESS and P-Trab groups, respectively (P = 0.681). Qualified success was achieved in 63% (n = 27) of patients in the P-Ex-PRESS group and 71% (n = 27) of patients in the P-Trab group (P = 0.561).By the criterion of pressure ≤18 mm Hg, 44% . In 12 patients (15.2%), BCVA remained at baseline after surgery and improved from one to nine lines in 61 (76.1%) patients. Vision deterioration due to posterior capsular-bag opacification was noted in one patient. Macular edema caused by chronic hypotension was observed in a single case during the follow-up.In the P-Trab group, BCVA decreased by one Snellen line in seven patients (8.8%), remained unchanged in six (7.7%), and increased by one to nine lines in 68 (83.5%) patients. The reasons for vision deterioration were posterior capsular-bag opacification, choroidal effusion, and dry age-related macular degeneration.P = 0.216). The average dose of 5-FU was 7.0 ± 3.5 mg in the P-Ex-PRESS group (a mean of 1.4 injections per patient) and 8.5 ± 2.3 mg in the P-Trab group (a mean of 1.7 injections) (P = 0.105). Needling was used in 11 patients (23.9%) in the P-Ex-PRESS group and in 12 in the P-Trab (30.8%) group (P = 0.305). One patient from the P-Ex-PRESS group (2.1%) and two patients from the P-Trab group (5.1%) (P = 0.462) underwent laser suturolysis. One patient from the P-Ex-PRESS group (2.3%) was fitted with an additional sealing suture (P = 0.354). Two patients from the P-Ex-PRESS group (4.6%) underwent reoperation: one due to extrusion of the mini seton through the scleral flap, and the other due to fibrosis of the filtering bleb. In both cases, classical trabeculectomy was performed. These patients were excluded from the study after the additional surgery, but their previous results were not excluded from the database. One patient from the P-Trab group (2.6%) underwent reoperation due to unsatisfactory IOP regulation. Two patients from the P-ExPress group (4.3%) and one (2.6%) from the P-Trab group had symptoms of malignant glaucoma that occurred at different time-points after surgery and were managed successfully with cycloplegics and compression dressing. The exact rate of complications is shown in Subconjunctival 5-FU injections were administered to nine patients (19.6%) in the P-Ex-PRESS group and nine patients (23.1%) in the P-Trab group . In the analyzed P-Trab group, mean astigmatism was 0.86 D before surgery and increased to 1.26 D after surgery (P > 0.05). Both before and after surgery, the direction of astigmatism was oblique.In this study, astigmatism was of the ATR type in the P-Ex-PRESS group, and the direction had not changed by the 6-month follow-up. Astigmatism values changed slightly during the follow-up period, dropping from an average of 1.15 D to 0.89 D , but the curvature of the posterior cornea also changed (0.4 to 0.9). However, the changes were not statistically significant after 3 months. They observed a non-significant correlation between the increase in astigmatism and the presence of a low IOP due to anti-glaucoma surgery, which was also previously reported by Razeghinejad [To date, the results of previous studies on the magnitude and direction of SIA are inconsistent. Hammel et al.  analyzedghinejad . Hornovághinejad . ClaridgLaw et al. compared the refractive results after P-Trab and phaco alone . No diffCunliffe et al. performed tests on 19 eyes after trabeculectomy and evaluated the refraction and keratometry data . Their fOur study has a few limitations. First, the data used for calculations were from autorefractometry and represented whole astigmatism, including lenticular astigmatism preoperatively and IOL postoperatively. In contrast, clinicians are concerned about overall astigmatism because it affects postoperative visual acuity. Second, a comparative study with keratometry could not be conducted, because the keratometry data were incomplete. Third, staff that performed the follow-up visits was not blinded; BCVA and IOP were measured with knowledge of the surgery that had been performed. Another limitation is the 6-month follow-up time. Existing data on astigmatism after anti-glaucoma surgery is inconsistent. According to one study, changes in corneal curvature after surgery can last up to 12 months , and othIn future studies, blinding of all investigators could improve the reliability of the outcome data. Also, cornea mapping should be obtained before and after surgery to permit an exact assessment of the astigmatism. Another issue for future investigations is assessment of the possible impact of the Ex-PRESS device (among the other implants) on endothelial cell loss.In conclusion, the P-Ex-PRESS and P-Trab procedures did not differ in inducing postoperative astigmatism or refractive errors; the astigmatism trend noted during observation remained the same in both groups (ATR in P-Ex-PRESS and oblique in P-Trab). Comparable results were found in the groups for final visual acuity. The two procedures showed similar antihypertensive efficacy in treating open-angle glaucoma. This study confirms the noninferiority of the Ex-PRESS device, which is relevant when making optimal clinical decisions with patients, as Ex-PRESS is comparatively less invasive than trabeculectomy.


We also explored the neurochemical identity of GHSRlPBN cells. To silence GHSRlPBN cells, Ghsr-IRES-Cre male mice were bilaterally injected intra-lPBN with a Cre-dependent viral vector expressing tetanus toxin-light chain. Unlike control wild-type littermates that significantly increased in body weight on the obesogenic diet , the heterozygous mice with silenced GHSRlPBN cells were resistant to diet-induced weight gain with significantly lower food intake and fat weight. The lean phenotype appeared to result from a decreased food intake compared to controls and caloric efficiency was unaltered. Additionally, silencing the GHSRlPBN cells altered food choice, significantly reducing palatable food consumption. RNAscope and immunohistochemical studies of the lPBN revealed considerable co-expression of GHSR with glutamate and pituitary adenylate cyclase-activating peptide (PACAP), and much less with neurotensin, substance P and CGRP. Thus, the GHSRlPBN cells are important for diet-induced weight gain and adiposity, as well as in the regulation of food intake and food choice. Most GHSRlPBN cells were found to be glutamatergic and the majority (76%) do not belong to the well-characterized anorexigenic CGRP cell population.The lateral parabrachial nucleus (lPBN), located in the pons, is a well-recognized anorexigenic center harboring, amongst others, the calcitonin gene-related peptide (CGRP)-expressing neurons that play a key role. The receptor for the orexigenic hormone ghrelin  is also abundantly expressed in the lPBN and ghrelin delivery to this site has recently been shown to increase food intake and alter food choice. Here we sought to explore whether GHSR-expressing cells in the lPBN (GHSR The parabrachial nucleus (PBN), located in the pons, is known to relay a broad range of sensory information to forebrain regions , includivia Y1 receptors) to increase food intake (There are indications that the lPBN forms part of the neurocircuitry engaged by (circulating) appetite-regulating hormones. Receptors for anorexigenic peptides such as leptin and glucagon-like peptide 1 (GLP-1) are expressed in the lPBN  and locad intake .in situ hybridization studies (Ghsr-IRES-Cre mice) crossed onto two different reporter strains  is abundantly expressed in the lPBN, revealed by  studies . More re strains . The GHS strains  as well rains . There is, however, considerable overlap and redundancy in systems ensuring sufficient food intake to sustain energy balance and it is therefore unclear whether selective silencing of GHSRlPBN cells would be sufficient to reduce food intake and limit body weight gain. Arguably, food choice may be less critical than food intake for survival, at least in the short term, and there may be less need for overlap and redundancy in the pathways regulating it. Therefore, our main hypothesis was that GHSRlPBN cells may play an important role in food choice. To silence GHSRlPBN cells, we used Ghsr-IRES-Cre mice  and hypothesized that they likely include one or more of the aforementioned lPBN cell groups.In the present study, we first sought to explore the impact of loss of GHSR signaling selectively in the lPBN on body weight progression, food intake and food choice. Based on ghrelin injection studies , silenciCre mice  which weCre mice  to blockCre mice  of the GGhsr-IRES-Cre heterozygous mice crossed (or not) with ROSA26-ZsGreen reporter mice B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J  as previously described  and water unless otherwise stated. All procedures were approved by the local Ethics Committee for Animal Care and Use at the University of Gothenburg (permit number: 27-2015 and 132-2016) and conducted in accordance with legal requirements of the European Community (Decree 86/609/EEC).Mice were housed in a 12-h light/dark cycle (lights on a 7:00) at 20 ± 2°C and 50% humidity and had Ghsr-IRES-Cre (Ghsr-Cre Het) male mice and 8 wild-type male littermates (WT) were injected bilaterally into the lPBN with a viral vector (AAV1-CBA-DIO-eGFP-Tetox-WPRE-pA)  but not in WT controls.Thirteen heterozygous WPRE-pA) . The expWPRE-pA) , is Cre-lPBN cells on sweet taste sensitivity.At 1 week after surgery, the individually housed mice were introduced to a high-fat, high-sugar (HFHS) free choice diet consisting of standard chow pellets, lard , 9% sucrose solution  and wateAt the end of the study, mice were perfused to verify the viral vector injection sites. Bilateral epididymal fat pads were collected and weighed prior to transcardial perfusion when mice were anesthetized.Two GHSR-silenced mice were excluded because post-mortem evaluation revealed that the injection site was off-target. Three GHSR-silenced mice were sacrificed 3 weeks post-surgery to monitor the viral vector expression and their data were included in the analysis of body weight. Another GHSR-silenced mouse showed a very strong body weight phenotype in response to Tetox and began to look weak and unhealthy 5 days before the end of the experiment. This mouse was therefore sacrificed at that time point and included in the analysis of body weight, food choice and saccharin preference. In addition, one control mouse died just after surgery. Thus, the final analysis included: (i) 8 GHSR-silenced mice and 7 control mice for the study of caloric intake, food choice and saccharin preference, (ii) 7–11 GHSR-silenced mice  and 7 control mice for the study of body weight development and caloric efficiency on the HFHS diet, and (iii) 7 GHSR-silenced mice and 7 control mice for the fat pad analysis.In an additional cohort of mice (6 GHSR-silenced and 9 controls) we performed a parallel study, measuring food intake and body weight progression in mice fed regular chow .Ghsr-IRES-Cre wild-type male mice and five Ghsr-IRES-Cre heterozygous male mice on the ZsGreen reporter background  were perfused to determine the neurochemical identity of the GHSRlPBN cells using fluorescent in situ hybridization (RNAscope) and immunohistochemistry, respectively (see sections below).In addition, four ®  and were deeply anesthetized by intraperitoneal (i.p.) injection of a combination of Sedastart vet®.  and Ketalar®  and placed in a stereotaxic frame. After exposure of the skull and application of a local anesthetic , two holes were drilled and the viral vector expressing Tetox was injected  into each side using a 31 gage stainless steel needle  connected via vinyl tubing to a Hamilton syringe placed in an infusion pump. The injection volume was optimized prior to the study in order to minimize spreading of the viral vector outside the lPBN. For injection of Tetox-expressing viral vector in the lPBN, the following coordinates were used: 5.34 mm posterior to bregma; 1.3 mm lateral to the midline; 3.7 mm ventral of the skull surface at bregma. After injection, the injection needle was kept in place for an additional 7 min and then slowly retracted to ensure full diffusion from the needle tip. After surgery, mice were injected with the sedation-reversing Sedastop vet®. , individually housed and allowed to recover for at least a week. Correct placement of the needle tip from the viral injections was confirmed post-mortem in all mice.All mice (26–41 g body weight) received a subcutaneous (s.c.) injection of the analgesic Rimadylad libitum access to a saccharin solution  in addition to water for 3 h/day on two consecutive days (the first day can be considered a habituation day) (The mice had ion day)  and 1 h/ion day) . The sac®. and Ketalar® mentioned above and perfused transcardially with heparinized 0.9% saline followed by 4% paraformaldehyde (PFA) in 0.1 M PB. The brains were dissected, post-fixed overnight at 4 °C in 4% PFA solution  and cryoprotected in 0.1 M PB containing 30% sucrose (25% sucrose for RNAscope) at 4°C until cryosection. Coronal sections containing the lPBN (30 μm-thick for immunohistochemistry or 14 μm-thick for RNAscope) were then cut using a cryostat and stored in tissue storage solution  at −20°C until further processing.The mice were deeply anaesthetized with the mixture of Sedastart vet2 , substance P and neurotensin emerged as appropriate candidates as a result of literature search , and com2 .in situ hybridization using RNAscope® was performed to study the potential co-expression of GHSR with glutamate (Slc17a6 probed), PACAP (Adcyap1 probed), substance P (Tac1 probed) and neurotensin in the lPBN of Ghsr-IRES-Cre wild-type male mice (n = 4). All reagents were purchased from Advanced Cell Diagnostics  if not stated otherwise.Fluorescent Ghsr probe (Cat. No. 426141-C3) contained 20 oligo pairs and targeted region 438–1385 (Acc. No. NM_177330.4) of the Ghsr transcript. The Slc17a6 (glutamate transporter) probe (Cat. No. 319171-C2) contained 20 oligo pairs and targeted region 1,986–2,998 (Acc. No. NM_080853.3) of the Slc17a6 transcript. The Adcyap1 (PACAP-coding gene) probe (Cat. No. 405911) contained 20 oligo pairs and targeted region 676–1,859 (Acc. No. NM_009625.2) of the Adcyap1 transcript. The Tac1 (substance P-coding gene) probe (Cat. No. 410351-C2) contained 15 oligo pairs and targeted region 20–1,034 (Acc. No. NM_009311.2) of the Tac1 transcript. The neurotensin probe (Cat. No. 420441) contained 20 oligo pairs and targeted region 2–1,188 (Acc. No. NM_024435.2) of the neurotensin transcript. Three-plex negative and three-plex positive control probes recognizing bacterial dihydrodipicolinate reductase, DapB (Cat. No. 320871) and PolR2A, cyclophilin and Ubiquitin (Cat. No. 320881), respectively, were processed in parallel with the target probes to ensure tissue RNA integrity and optimal assay performance.The ® Multiplex Fluorescent v2 Assay (Cat. No. 323100). The Adcyap1 and neurotensin probes were labeled with Opal 520 , the Ghsr probe with Opal 570 , and the Slc17a6 and Tac1 probes with Cy5 .All incubation steps were performed at 40°C using the ACD HybEz II hybridization system (Cat. No. 321462). On the day before the assay, every 6th section throughout the lPBN was mounted on SuperFrost Plus slides , dried at room temperature, briefly rinsed in autoclaved Milli-Q purified water, air-dried and baked at 60°C overnight. From one of the animals, sections from the same region of the brain were also mounted for use with the positive and negative control probes. On the day of the assay, slides were first incubated for 7 min in hydrogen peroxide (Cat. No. 322335), submerged in Target Retrieval (Cat. No. 322001) at a temperature of 98.5–99.5°C for 7 min, followed by two brief rinses in autoclaved Milli-Q purified water. The slides were quickly dehydrated in 100% ethanol and allowed to air-dry for 5 min. A hydrophobic barrier was then created around the sections using an ImmEdge hydrophobic barrier pen (Cat. No. 310018) and the sections were incubated with Protease Plus (Cat. No. 322331) for 30 min. The subsequent steps, i.e., hybridization of the probes and the amplification and detection steps, were performed according to the manufacturer’s protocol for the tyramide-based RNAscopeSections were counterstained with DAPI and coverslipped with Prolong Diamond Antifade mounting medium  and stored in the dark at 4 °C until image acquisition.lPBN cells overlap with the CGRP-expressing population, immunohistochemistry was performed on every 6th lPBN-containing sections from Ghsr-IRES-Cre heterozygous male mice on the ZsGreen reporter background (n = 5). The sections were incubated with goat anti-CGRP antibody  overnight at 4 °C followed by Alexa Fluor 594 chicken anti-goat secondary antibody  for 1 hr at room temperature before being mounted onto glass slides and coverslipped with ProLong Diamond Antifade mounting medium. The slides were stored at 4 °C until image acquisition.To determine whether the GHSRImages of the immunohistochemical staining for CGRP and the RNAscope for GHSR, glutamate, PACAP, substance P and neurotensin were acquired using a laser scanning confocal microscope  at the Centre for Cellular Imaging at Gothenburg University. A Plan-Apochromat 20x/0.8 (WD = 0.55 mm) objective was used with 3 × 2 tiling settings to image the immunohistochemical staining, while a Plan-Apochromat 40x/1.3 Oil DIC objective was used with 6 × 4 tiling and z-stack settings to image the RNAscope. We used the same image acquisition settings for all images in each study and ensured that these settings did not detect any signal in the negative control sections (sections incubated with the secondary antibody but not with the primary for immunohistochemistry and sections incubated with the negative control probe for RNAscope). The z-stack images were processed using the maximum intensity projection function in the Zen Black software (Zeiss). The final images were then stitched and the cells counted in ImageJ/Fiji . The cell counter plug-in was used to count positive cells and co-localization in the lPBN. DAPI was used to make sure any positive signal counted was indeed a cell, although we did not simultaneously check for neuronal/glial markers. Two and 4–5 lPBN sections from bregma −5.07 to −5.33 mm were used for the quantification of the immunohistochemical and the RNAscope images, respectively.post hoc test was used to adjust for multiple comparisons. The body weights at the start of exposure to the HFHS diet and after 23 days for each group separately were compared using a paired sample Student’s t-test. Total caloric intake and food choice data on HFHS free choice diet were analyzed using a one-way ANOVA (group). A Student’s t-test was used to examine data from the saccharin preference test as well as the fat pads weights. The results are reported as mean ± SEM and statistical significance was set at p < 0.05.Data were analyzed using IBM SPSS Statistics 25 . All data were tested for normal distribution using a Shapiro-Wilk test and for homogeneity of variances using a Levene’s test. Throughout the 23 days of HFHS free choice diet exposure, % body weight change and total energy intake were analyzed by one-way repeated measures ANOVA (group) with the days as the within-subject factor. Bonferroni t(6) = −3.20, p = 0.019]  = 6.06, p = 0.030] (p = 0.043) and on days 14–23   .F = 5.91, p = 0.032]  compared to controls (1.7 ± 0.3 g) [= 0.032] .lPBN cells to suppress food intake and decrease body weight gain could only be observed in mice fed a HFHS diet  = 22.0, p < 0.001)  = 15.0, p = 0.003) and the difference was significant at each time point from day 5 of the HFHS free choice diet until the last day of the study  . Total ehe study . The calhe study .Any trends toward a lowering in body weight data in the cohort fed regular chow are unlikely to be due to an increase in energy expenditure, since we did not detect any difference in caloric efficiency .F = 4.74, p = 0.049] while that of chow and water did not differ between groups  = 2.16, p = 0.166]  = 8.99, p = 0.010]  compared to controls  [n groups . Lard in= 0.166] . In line= 0.010] .GHSR-silenced and control mice had similar saccharin preference during the habituation day (Day 1)  and the two test days .The RNAscope study shows that, in the lPBN of mice, the majority of the GHSR-expressing cells  revealed that the majority of GHSRlPBN cells are glutamatergic (over 80%), just over 40% express PACAP, a quarter express substance P, just under a quarter express CGRP, while just under 10% express neurotensin.Recent studies using Cre mice  identifyf/f mice)  . Given t/f mice) , the expogenesis . By contlPBN cells consumed significantly less sucrose solution relative to control mice. We did not detect any significant reduction in lard intake, likely due to high variability between the mice and also the small number of animals studied. However, when we explored caloric intake of sucrose plus lard together, the difference of intake of these combined palatable foods reached a higher significance level than for sucrose alone, comparing mice with silenced GHSRlPBN cells and controls. Chow intake was not altered by silencing of GHSRlPBN cells, when offered in the choice diet or as a single food  is not diametrically opposite to that of central ghrelin injection in rats   versus a ghrelin injection (It would appear that the impact of silencing the GHSRof chow) . This is the VTA . Ghrelinnjection . TentatilPBN cells is not due to an impairment of sweet taste sensation. The role of the PBN as a relay for taste/gustatory information was established many decades ago , tested using a single saccharin concentration . Furtherades ago . Yet, thades ago  and in tades ago . The GHSlPBN cells in this way caused a phenotype identifies these cells as having a role in this phenotype. Arguably, we do not know if GHSR is the key signal in these cells for the effects observed, since the cells will no longer respond to any afferent signal. Activation by PYY of the Y1 receptor expressed in the lPBN was shown to also increase food intake, for example  , that is example . Yet, ev example . Additio example . Other p example , μ-opioi example  and cann example . It would leptin . Of thesd leptin .lPBN cells are a heterogeneous population within which the majority of the cells are glutamatergic, slightly less than half co-express PACAP and small proportions are substance P- and neurotensin-positive. Moreover, a surprising and interesting fact is that, GHSR is expressed on approximately 15% of the cells from each neuropeptide/neurotransmitter-expressing population studied . This might point toward an important role of GHSR in modulating different circuits in the lPBN. Regarding the potential circuitry of the GHSRlPBN cells, Niu and colleagues identified glutamatergic lPBN neurons that project to orexin-expressing hypothalamic neurons in rats  that the GHSR in rats  that are in rats . In addi in rats , 2017. T in rats . The preGhsr-IRES-Cre mice. We found that the GHSR- and CGRP-expressing cells of the lPBN are mostly separate populations, but that a quarter of the GHSR-expressing cells also express CGRP. Thus, the orexigenic GHSRlPBN cells and the well-known anorexigenic circuit in the lPBN are mainly distinct, but a few cells seem to be common to both. Interestingly, CGRP has been shown to be expressed on at least some of the PACAPlPBN neurons projecting to the CeA and BNST .The work was undertaken in the group of SD, who is the lead scientist of this study. ML performed the experiments and analyzed the data with assistance from FP-S, IS, ES, and TB. ML, FP-S, RA, and SD designed the studies. ML wrote the manuscript with guidance from SD and FP-S. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Malnutrition is the main risk factor for most common communicable diseases. The aim of this study is to determine the relationship between country-level prevalence of early childhood caries (ECC), malnutrition and anemia in infants and preschool children.Matched country-level ECC, malnutrition and anemia prevalence were generated from databases covering the period 2000 to 2017. Multivariate general linear models were developed to assess the relationship between outcome variables  and the explanatory variable  adjusted for gross national income per capita. Adjusted regression coefficients (B) and partial eta squared were computed.P = 0.03). In 3–5-year-olds, the strongest and only significant association was between the prevalence of ECC and anemia (η2 = 0.08): 1 % higher prevalence of ECC was associated with 0.14% lower prevalence of anemia .The mean (standard deviation (SD)) ECC prevalence was 23.8 (14.8)% for 0–2 year-olds and 57.3 (22.4)% for 3–5-year-olds. The mean (SD) prevalence of wasting was 6.3 (4.8)%, overweight 7.2 (4.9)%, stunting 24.3 (13.5)%, and anemia 37.8 (18.1)%. For 0–2-year-olds, the strongest and only significant association was between the prevalence of ECC and overweight (η2 = 0.21): 1 % higher ECC prevalence was associated with 0.12% higher prevalence of overweight (B = 0.12, Country-level prevalence of ECC was associated with malnutrition in 0–2-year-olds and with anemia in 3–5-year-olds. The pathway for the direct relationship between ECC and overweight may be diet related. The pathway for the inverse relationship between ECC and anemia is less clear and needs further investigations. There are global concerns about the multi-facet impact of malnutrition in children. The term malnutrition includes undernutrition , which is present most often in disadvantaged communities, and over consumption of food (overweight and obesity), which is present in both developed and developing communities . MalnutrEarly childhood caries (ECC) is dental decay that affects the primary dentition in children younger than 6 years of age . It is aRecent studies have highlighted the relationship between ECC and malnutrition , micronuThe purpose of this study was to determine the association between country-level prevalence of ECC with malnutrition and anemia in infants and preschool children. The null hypothesis of the study was that ECC prevalence in 0 to 2-year-olds and 3 to 5-year-olds is not associated with malnutrition or anemia.This was an ecologic study. We collected macro-level data about ECC, anemia and nutritional status of children under six years of age for the period January 2007 to October 2017 for United Nations member States .According to the American Academy of Pediatric Dentistry, children < 72 months of age with one or more decayed, missing due to decay or filled primary tooth surfaces have ECC . The datStunting: below minus two SDs from median height-for-age.Wasting: below minus two SDs from median weight-for-height.Overweight: above one SD from median weight-for-height.Information on nutritional status was obtained from country-level data jointly produced by the WHO, UNICEF and World Bank in 2018 , 28 coveThe prevalence of wasting, stunting and overweight were reported as the percentage of children 0–5 years old who met the definition.We used country-level estimates for anemia prevalence from the WHO , where ip values were calculated. Multivariate analysis of variance (MANOVA) with the SPSS procedure multivariate general linear analysis was used to develop two separate models assessing the relationship between outcome variables  and two explanatory variables  for each two-age group. Each model was adjusted for the economic level of the country according to the 2017 Gross National Income per capita calculated with the World Bank Atlas method [p values and partial eta squared (η2 as measure of effect size) were computed. Residual plots were assessed for the randomness of residuals’ distribution to ensure that model assumptions apply. Variance inflation factors were calculated to assess collinearity. Significance level was set at 5%. Statistical analyses were performed with SPSS version 22.0 .The data sets  were matched by country. Scatter plots were used to represent the correlation between the prevalence of anemia, malnutrition and ECC in the two age groups (0–2-year-olds and 3–5-year-olds), and correlation coefficients and s method  based ons method . The groECC data were available for 39 countries for 0–2-year-olds and for 86 countries for 3–5-year-olds. Data on malnutrition and anemia were available for 128–185 countries, depending on malnutrition type. Combined ECC, anemia and malnutrition data were available for 26 countries in the age 0–2-years-old and 55 countries in the age 3–5-years-old (See Supplement The mean (SD) ECC prevalence was 23.8 (14.8)% in 0–2-year-old children and 57.3 (22.4)% in 3–5-year-old children. The overall mean (SD) prevalence of wasting was 6.3 (4.8)%, of overweight was 7.2 (4.9)%, of stunting was 24.3 (13.5)%, and of anemia was 37.8 (18.1)%.The 26 countries that had complete data on ECC, anemia and malnutrition for 0–2-year-olds were two (7.7%) low-income countries, 10 (38.5%) lower middle-income countries, nine (34.6%) upper middle-income countries, and five (19.2%) high-income countries. The 55 countries that had complete data on ECC, anemia and malnutrition for 3–5-year-olds were six (10.9%) low-income countries, 19 (34.5%) lower middle-income countries, 22 (40%) upper middle-income countries, and eight (14.5%) high-income countries.P = 0.40), stunting , and anemia . The prevalence of overweight was positively, moderately and significantly correlated with the prevalence of ECC .Figure P = 0.29), and overweight . However, the prevalence of anemia was positively, weakly and significantly correlated with the prevalence of ECC .Figure P = 0.03). For children 3–5 years of age, the greatest effect size and only significant association was between the prevalence of ECC and the prevalence of anemia (η2 = 0.08), where countries with 1 % higher prevalence of ECC had a 0.14% lower prevalence of anemia . No collinearity was observed (variance inflation factors < 5).Results of the multivariate general linear analysis controlled for economic level are reported in Table To our knowledge, this study provides the first evidence on the relationship between country-level prevalence of ECC, malnutrition, and anemia. However, not all types of malnutrition were associated with ECC. We found a positive and significant relationship between country-level prevalence of overweight and ECC in children 0–2-years of age, with higher prevalence of overweight associated with higher ECC prevalence. There was also an inverse relationship between country-level prevalence of anemia and ECC in 3–5-years olds with lower prevalence of anemia in countries with higher prevalence of ECC.Overweight was associated with ECC only in children ≤2-years of age. The relationship between ECC, and childhood growth and development is not clear , 31. TheThe higher prevalence of overweight in countries with higher estimates of ECC in 0–2-year-olds may reflect the findings by El Tantawi et al.  who repoThe few studies that have assessed the relationship between ECC and nutritional status often included age ranges larger than that of ECC. Four studies conducted amongst preschool children, found no association between ECC and overweight in children 3 years of age , 2–5-yeaAnemia, which is a complication of malnutrition and factors not malnutrition related , 36, wasThe present findings may have implications at the micro-level. Malnutrition is a complex disorder not solely caused by lack of food . FeedingOne of the strengths of this study is that collated data on malnutrition included high-quality data from the Demographic Health Survey . HoweverA limitation of this study was use of the World Health Organization’s criteria for assessing caries by many of the epidemiological surveys . This asCountry-level prevalence of ECC was associated with malnutrition in 0–2-year-olds and with anemia in 3–5-year-olds. We also observed age-related disparities in the relationship between ECC, malnutrition, and anemia. The disparities reiterate the need for future analysis of ECC-related data for the two age groups as earlier highlighted by El Tantawi et al. . FurtherAdditional file 1. Countries with complete data on early childhood caries, anemia and malnutrition.


Conus for the treatment of neuropathic pain. α-Conotoxin RgIA and a potent analog, RgIA4, have previously been shown to prevent the development of neuropathy resulting from the administration of oxaliplatin, a platinum-based antineoplastic drug. Here, we have examined its efficacy against paclitaxel, a chemotherapeutic drug that works by a mechanism of action distinct from that of oxaliplatin. Paclitaxel was administered at 2 mg/kg ) every other day for a total of 8 mg/kg. Sprague Dawley rats that were co-administered RgIA4 at 80 µg/kg (subcutaneously (SC)) once daily, five times per week, for three weeks showed significant recovery from mechanical allodynia by day 31. Notably, the therapeutic effects reached significance 12 days after the last administration of RgIA4, which is suggestive of a rescue mechanism. These findings support the effects of RgIA4 in multiple chemotherapeutic models and the investigation of α9α10 nicotinic acetylcholine receptors (nAChRs) as a non-opioid target in the treatment of chronic pain.Chemotherapeutic drugs are widely utilized in the treatment of human cancers. Painful chemotherapy-induced neuropathy is a common, debilitating, and dose-limiting side effect for which there is currently no effective treatment. Previous studies have demonstrated the potential utility of peptides from the marine snail from the genus  Taxus brevifolia), and perhaps the most well-known natural-product chemotherapeutic. It is used as a first-line treatment for breast, ovarian, and non-small cell lung cancers dec-2-ene sesquifumarate ((R)-ICH3) or PNU-282987, positive allosteric modulation with GAT107 or PNU-120596, and silent agonism with (R)-N-(4-Methoxyphenyl)-2-((pyridin-3-yloxy)methyl)pi-perazine-1-carboxamide Dihydrochloride (R-47) have shown efficacy in models of inflammatory and neuropathic pain ,76,77,78 effects ,80. ABT-While chemotherapies are diverse in their mechanisms of action, several drugs across different families have produced similar neuropathies. The use of natural products as a source of therapeutics has proven to be effective but with caveats. Harnessing the evolutionary refinement of these compounds has provided a rich collection of highly specific pharmacological tools. The discovery of paclitaxel was a fortuitous hit from a concerted screening of natural products coordinated by the United States National Cancer Institute looking specifically for compounds that would be efficacious against cancers . AlthougMale Sprague Dawley rats (Envigo) weighing 200–300 g were used in all rat experiments. Rats were housed in groups of two rats per cage with food and water available ad libitum. Animals were maintained in a temperature- and humidity-controlled animal colony maintained on a 12-h light/dark cycle (lights on at 07:00). Male C57BL/6J mice (Jackson Laboratories) were used in all mouse experiments. Mice were housed in groups of four or five per cage with food and water available ad libitum and on the same light cycle. All rat studies were conducted under the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of New England, protocol number 042617-001. All mice experiments were conducted under the approval of the University of Utah IACUC, protocol number 17-08002. The Animal Care and Use Programs and Facilities of the University of Utah are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.A clinical formulation of paclitaxel was employed. Paclitaxel USP, extracted from Taxus X media ‘Hicksii’ , came in solution at a concentration of 6 mg/mL. The vehicle used for dilution consisted of 1 part 1:1 Cremophore:EtOH mixture and 2 parts 0.9% saline, and 2 mg/mL sodium citrate. Rats were given a dose of 2 mg/kg (IP) every two days for a total of 4 doses, at a volume of 1 mL/kg body weight. RgIA4 was dissolved in 0.9% saline and injected (SC) once per day, 5 days a week for a total of 15 doses at 1 mL/kg body weight for rats and 4 mL/kg body weight for mice . Upon con = 8 unless otherwise specified. Animals were placed in a clear Plexiglass chamber and allowed to habituate for 15–60 min. Touch-Test filaments  were used for all testing. For rats, the 2.0 g (4.31) filament was used to start. Clear paw withdrawal, shaking or licking was considered a positive or painful response. This up-down method was stopped four measures after the first positive response. The withdrawal threshold was calculated using the up-down Excel program generously provided by Dr. Michael Ossipov . The filament range for rats was 3.61, 3.84, 4.08, 4.31, 4.56, 4.74, 4.93, 5.18.Tactile allodynia was quantified by measuring the hindpaw withdrawal threshold to Von Frey filament stimulation, using the up-down method previously reported ,82. Thron = 8 unless otherwise specified. Briefly, animals were placed in a clear Plexiglass chamber and allowed to habituate for 15–60 min. A radiant heat source was then focused on the plantar surface of the hindpaw with the paw withdrawal time automatically determined. The intensity of the heat source was adjusted so that baseline latency was approximately 15 s. A test cutoff time of 30 s was observed to avoid tissue damage. Thermal allodynia was quantified by measuring the hindpaw withdrawal latency to noxious radiant heat application in unrestrained animals using the plantar test apparatus (rats: Ugo Basile), as previously reported . Throughn = 8 unless otherwise specified. A hot/cold plate (Ugo Basile) was used and cooled to 5 °C. To help achieve reliable results a layer of distilled water coated the cold plate. Each rat was placed on the plate, one at a time. The time it took the rat to lick or flick a hind paw or jump to escape the plate was recorded to the nearest 0.1 s. A 5 min cutoff time was used to remove the rat from the plate if they had not yet shown a response to avoid tissue damage.Rats underwent cold plate testing to determine if any sensitivity to cold developed following paclitaxel injection. Throughout the study, experimenters were blinded to the identity of the injected compound. Rats were divided into treatment groups of t-test or two-way ANOVA was performed followed by a Bonferroni posttest using GraphPad Prism software version 6.07 for Windows, GraphPad Software, La Jolla, CA, USA. Significance was established at the p < 0.05 level. In the rat assays, dose- and time-response curves were constructed for each pain model. Mean and standard errors of the mean were calculated. To determine statistical significance either an unpaired


Structurally-diversified bile acids (BAs) are involved in shaping of intestinal microbiota as well as absorption of dietary lipids. Taurocholic acid, a conjugated form of BA, has been reported to be a factor triggering germination of a wide range of spore-forming bacteria in intestine. To test a hypothesis that other BAs also promote germination of intestinal bacteria, we attempted culture of bacteria from ethanol-treated feces by using a series of BAs. It was found that conjugated-BAs, notably three glycine-conjugated BAs, glycodeoxycholic acid and glycochenodeoxycholic acid, significantly increased the number and the species variety of colonies formed on the agar plate. These colonized bacteria mostly belonged to class Clostridia, mainly consisting of families Lachnospiraceae, Clostridiaceae, and Peptostreptococcaceae. There were several types of bacteria associated with different sensitivity to each BA. Eventually, we isolated 72 bacterial species of which 61 are known and 11 novel. These results demonstrate that the culturable range of bacteria in intestine can be widened using the germination-inducing activity of BAs. This approach would advance the research on spore-forming Clostridia that contains important but difficult-to-cultured bacteria associate with host health and diseases. The number of bacterial cells residing in the body of human is estimated to be approximately 40-trillion bacterial cells2, consisting of several hundreds of bacterial species out of total 4,500 estimated for all human on the earth3. More than 2,600 of 4,500 species are estimated to be uncultured and have never been isolated in the laboratory4, despite including members that are believed to play an important biological role that remains undiscovered5. Although several metagenomic studies shed light on these uncultured bacteria that may have avoided past culture strategies6, a new wave of gut microbiota culturomics is decreasing the number of unclassified microbial diversity within the gut ecosystem12.The crosstalks of host-gut microbes interaction plays a crucial role in the health and disease of human being, accounts for approximately half of the gut microbial community, many of which are known as spore-forming bacteria14. The high tolerance of these spore-forming bacteria to gut environment stress such as high and low temperature, nutrient and oxygen and drug attack allows their long-term survival16. It is also known that spores tolerant to the outside gut environment facilitate the host-to-host transmission of bacteria through spatio-temporal means, which is not limited to pathogens, but includes commensals beneficial to health14.The phylum Firmicutes, which includes Lachnospiraceae, Ruminococcaceae and Clostridiaceae18. BAs are mostly reabsorbed and recycled at the end of the small intestine, and thereafter parts of them passed through to the large intestine, after which they are processed to secondary BAs by large intestinal microbiota. Specifically, conjugated BAs are deconjugated by the function of the bacterial bile salt hydrolase (BSH)20 and then converted to secondary BAs through 7α-dehydroxylation, resulting in deoxycholic acids (DCA) and lithocholic acid (LCA) from CA and CDCA respectively. Alternatively, 7α-epimerization results in ursodeoxycholic acid (UDCA) from CDCA23. Since these BA converting enzymes are unique to certain species of bacteria, the final composition of BAs will differ depending on the composition of gastro-intestinal microbiota24.Bile stress is one of the stresses experienced by intestinal bacteria. In humans, there are two main BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), which are produced and secreted into the small intestine as taurine- or glycine-conjugated forms to aid lipid absorption by forming water-soluble micelles25. In contrast, some spore-forming bacteria including pathogens and commensals, germinate in response to BAs in the intestinal tract7. The germination of Clostridioides difficile spores, the pathogen that causes diarrhea, is stimulated by a combination of nutrients and bile salts present in the gastrointestinal tract, specifically taurocholic acid (TCA)29. It was recently reported that TCA also induces germination of certain populations of spore-forming bacteria in the intestine and expands the culturability of gut bacteria7.With bactericidal activity, BAs provide selective pressure for the colonization of bile-sensitive bacteriaHere, we hypothesize that many spore-forming bacteria in intestinal tract control germination by using TCA as the environmental signal. To address this hypothesize, we demonstrated the germination-inducing activity of a series of human BAs against spore-formers in feces, which are expected to include a number of yet-to-be cultured bacterial species.p < 0.05, Wilcoxon signed-rank test). GCDCA and GDCA in particular, showed approximately two-fold higher activity compared to TCA, which is a known germination inducer, although not statistically significant .To examine the activity of a series of BAs on spore germination, seven fresh fecal samples were treated with an equal volume of 70% (v/v) ethanol to kill vegetative cells. Surviving spores were treated with each BA then spread and culture on GAM agar medium. For a negative control, non-BA treated spores were also spread and culture on the plate in the same manner. The number of colonies formed from samples treated by each BA signatures was counted individually for the seven fecal samples and statistically compared with the different BA groups Table . The colTo assess the population and the phylogenetic variety of intestinal bacteria responsive to the 15 BAs, the bacterial cells of colonies formed on each BA plate were collected and subjected to 16S rRNA gene amplicon sequencing. For the control, colonies from fecal samples without ethanol treatments, which represented cultivable vegetative cells of non-spore-forming and spore-forming bacteria, were analyzed in the same manner. The relative abundance of each bacterial family was determined for each plate. The data obtained from fecal samples of 7 donors were statistically compared among each BA-treated groups and the non-ethanol treated control group Fig. . Neither30.The heat map in Fig. We cultured 438 bacterial colonies isolated from the ethanol-BA treated fecal samples of seven donors, as well as 67 colonies from untreated feces of the same donors Table . Theses 31. Consequently, 81 known species were identified, while 11 OTUs, all of which were uniquely isolated from the ethanol-BA treated samples, were not identified to any known species in the database. Further, of the 11 OTUs, 3 were recognized as novel candidate genus. These candidates belong to either the Lachnospiraceae or Clostridiaceae families of the Firmicutes phylum.Each OTU was taxonomically annotated by comparison against the EzBioCloud 16S rRNA gene database. Consequently, 94 OTUs were classified into four main phyla of human intestinal bacteria, namely Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria , each with 2 ml RNA later for 16S rRNA gene sequencing analysis or phosphate buffered saline (PBS) for bacterial culture. Collected samples in RNAlater were stored at 4 °C until further processing for DNA extraction. Collected samples in PBS were immediately processed within 1 h to preserve the viability of anaerobic bacteria.The BAs used in this study are: CA , CDCA , UDCA , DCA , LCA , their taurine conjugated forms, TCA , TCDCA , taurodeoxycholic acid , taurolithocholic acid , tauroursodeoxycholic acid , and their glycine conjugated forms, GCA , GCDCA , GDCA , glycolithocholic acid , glycoursodeoxycholic acid . Stock solution of BAs was prepared by dissolving each BA in dimethyl sulfoxide to be 10% (w/v) except for TLCA, GLCA, TDCA, and GDCA to be 1% (w/v) followed by filter sterilization. Immediately before use, the BA solutions were diluted in reduced PBS to be 0.1% (w/v) except 0.01% (w/v) for the four BAs.7. Thereafter, the material was washed three times with PBS. Then, the ethanol-treated feces were resuspended with BA solutions (0.1 or 0.01 mg BAs per ml PBS) in order to stimulate spore germination and a spread on agar plate with Gifu-anaerobic medium . For ethanol-untreated control, the fecal suspension was serial dilution and spread on the agar plates with GAM.Bacteria culturing was performed under anoxic conditions in an anaerobic chamber . The fecal samples were homogenized in PBS (0.1 g stool per ml PBS), added by an equal volume of 70% (v/v) ethanol, and then incubated for 4 h at room temperature under ambient aerobic condition in order to kill vegetative cellsThe inoculated agar plates were incubated for 72 h at 37 °C in the anaerobic chamber. The colonies formed on the agar medium were picked and inoculated in 3 ml GAM broth, and thereafter incubated overnight for the liquid culture. The cultured bacteria were reserved in glycerol stock at − 80 °C while analyzing the sequence of full-length 16S rRNA gene.39. Bacterial DNA was also extracted from fecal samples using the same DNA extraction process. The extracted DNA was stored at − 30 °C until sequencing was performed on the Illumina MiSeq.All colonies on the plates were picked and suspended in 1 ml PBS by using inoculating loop. The genomic DNA was extracted from the pool by the beads-phenol method as previously described39. PCR products were pooled and combined in equimolar amounts for sequencing using the Illumina MiSeq platform, generating 300 bp paired-end reads.The variable region, V3-V4, of the 16S rRNA gene, was amplified from the extracted DNA as a template using the universal primers 341f. (5′- CGCTCTTCCGATCTCTGCCTACGGGNGGCWGCAG -3′) and 805r (5′- TGCTCTTCCGATCTGACGACTACHVGGGTATCTAATCC -3′), and TaKaRa Ex Taq HS (Takara Bio). The amplified products were then used as templates in a second PCR for further amplification using barcode-tagged primers. Both PCRs were performed as described previouslyhttps://drive5.com/useach/manual/upp_ill_pe.html).40Firstly, the pair of sequence reads were merged using the fastq_mergepairs script with mismatched windows up to 20 bases. After quality filtering, dereplication, the discard of singletons and length trimming, the merged sequences were clustered into 286 OTUs, each representing greater than 97% identity, using the UPARSE-OTU algorithm. The taxonomy of each OTU was assigned at phylum to species level using the EzBioCloud 16S rRNA gene database and the VSEARCH program41 and a cut-off of 0.97.The sequences obtained from feces and cultured bacteria were processed using the Uparse pipeline in Usearch version 9.2 and 10.0.  and then analyzed using the neighbor joining method42. For the neighbor-joining tree, evolutional distances were calculated based on a two-parameter model43. The rarefaction analysis was calculated by using the Usearch alpha_rare command.The taxonomy of the cultured isolates was analyzed using full-length 16S rRNA gene sequences. The full-length 16S rRNA gene fragment was amplified by PCR using the 27F (5′- AGAGTTTGATCCTGGCTCAG -3′) forward and 1497R (5′- GGTTACCTTGTTACGACTT -3′) reverse primers. Purified PCR products were subjected to Sanger sequencing by GENEWIZ . The resulting sequences were aligned using the Uparse/Usearch pipeline and were then grouped into OTUs, each representing greater than 98.7% sequence identity as per the UPARSE-OTU algorithm. The full-length 16S rRNA gene sequence of each species-level OTU was searched in the EzBioCloud 16S rRNA gene database  to assign taxonomic designations to the species level and either known or novel candidate species statushttps://www.r-project.org/) and Excel 2011 (Microsoft). For comparing cfu among sample groups, the paired Wilcoxon singled-rank test was used. Cultured bacteria were clustered based on the relative abundance of OTUs cultured with each BA, in which hierarchical clustering was calculated based on complete linkage of the Eucidean distances matrix by the Heatmap.2 function in the R gplots package.Statistical analyses were performed using the R package (V.3.2.2) (Raw sequence data were deposited in the DNA Data Bank of Japan (DDBJ) sequence read archive (DRA010323) under BioProject no. PRJDB012698, which contains links and access to stool sampling and cultured data under BioSample SAMD00230455 to SAMD00230551.This study was approved by the Ethics Committees of the Faculty of Agriculture, Kyushu University (No. 19-006). All methodswere carried out in accordance with the relevant guidelines and regulations. Written informed consent was obtained from donors of fecal samples. We entered and analyzed all samples anonymously and will publish all data anonymously using personal numbers.Supplementary Information.


Upfront genetics/cytogenetics and minimal measurable disease (MRD) are becoming relevant biomarkers in the process of post-remission transplant allocation in AML. However, until recently the transplantation choice relied on the availability of a fully matched familiar donor, whereas individual patient- and disease-related characteristics played a secondary role in transplant allocation. In this paper we analyzed the evolution of the transplantation policy at our center in a 20-year time interval. At the beginning of our observation patients were submitted to allogeneic transplant, per protocol, mostly if a fully matched family donor was available or to autologous transplant if no fully matched family donor was identified (“donor vs. no donor” strategy) regardless of upfront genetics/cytogenetics or MRD status. Thereafter, persistence of MRD after consolidation cycle was included in the decision-making process for transplant selection. Patients with favorable and intermediate-risk cytogenetic risk were to receive allogeneic or autologous stem cell transplantation if MRD positive or negative, respectively, (“transplant vs. no transplant” strategy). In this cohort, patients with FLT3-ITD or adverse risk karyotype were submitted to allogeneic transplant as well. p = 0.012 and 42.0% vs. 19.5%, p = 0.0003). The favorable impact of this MRD-driven strategy was evident for the intermediate-risk category, particularly for MRD positive patients. In the low-risk category, the significantly lower CIR of the MRD-driven cohort did not translate into a survival advantage. In conclusion, a MRD-driven transplant allocation may play a better role than the one based on the simple donor availability. This approach determines a superior outcome of intermediate-risk patients whereat in low-risk ones a careful evaluation is needed for transplant allocation.Measurable residual disease (MRD) is increasingly employed as a biomarker of quality of complete remission (CR) in intensively treated acute myeloid leukemia (AML) patients. We evaluated if a MRD-driven transplant policy improved outcome as compared to a policy solely relying on a familiar donor availability. High-risk patients  received allogeneic hematopoietic cell transplant  whereas for intermediate and low risk ones (CBF-AML and NPM1-mutated), alloHCT or autologous SCT was delivered depending on the post-consolidation measurable residual disease (MRD) status, as assessed by flow cytometry. For comparison, we analyzed a matched historical cohort of patients in whom alloHCT was delivered based on the sole availability of a matched sibling donor. Ten-years overall and disease-free survival were longer in the MRD-driven cohort as compared to the historical cohort (47.7% vs. 28.7%,  Nowadays, allogeneic hematopoietic cell transplantation  represents the most effective antileukemic treatment and it is increasingly employed in patients affected with acute myeloid leukemia (AML) up to the age of 70 yrs . In spitMeta-analyses of large prospective studies indicate that, whatever the underlying genetic/cytogenetic abnormality, the beneficial effect of alloHCT takes place as soon as the risk of relapse exceeds 35–40%; when probabilities of relapse are below those percentages the risk of non-relapse mortality (NRM) will attenuate the survival advantage of alloHCT .Nevertheless, the increasing knowledge of the genetic landscape of AML has shed light on the complex gene interplay affecting prognosis in AML and some disease categories  have resulted to be more complex than expected with a final outcome that may eventually be not as favorable as expected . If thesIn this view, measurable  residual disease (MRD) promises to be a robust biomarker in that it captures the diversities of the different genetic/cytogenetic features of AML and recapitulates other patient-specific heterogeneities regarding drug bioavailability, metabolism and chemoresistance ,10. OnceWe evaluated if MRD positivity after consolidation might allow to identify patients with high-risk of disease recurrence among those without favorable or intermediate genetics/cytogenetics (“MRD-driven” strategy) and if an MRD-driven alloHCT in first CR might lead to overall (OS) and disease-free survival (DFS) improvement in these categories of patients. Establishing a proper transplant policy was a crucial part of the MRD-driven approach. In fact, once declared at high-risk, patients were to receive alloHCT whatever the source of stem cells: matched sibling donor (MSD), matched unrelated donor (MUD), umbilical cord blood (UCB) or haploidentical related donor (HRD). Well aware of the potential biases deriving from an historical comparison between populations of patients spanning over a 20 years period, we analyzed the outcome of a matched cohort of patients who had in common the same treatment design except being submitted to alloHCT only if a fully matched family donor was available or to autologous stem cell transplant (AuSCT) if not (“donor-driven” strategy), regardless of the baseline risk profile or of the MRD status. We analyzed a population of 185 AML consecutive patients affected with de novo non-M3 AML in CR1, diagnosed in the period between 1998 and July 2013 at Tor Vergata University and St. Eugenio Hospital of Rome. In our retrospective analysis, post-consolidation timepoint was identified as the most informative one for MRD prognostication in European Organization for Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto (GIMEMA) protocols . Patient2 as intravenous bolus followed by SDAC 100 mg/m2 given as a daily continuous infusion for 10 days; i.v. etoposide 100 mg/m2/d on days 1 to 5; and on days 1, 3, and 5 one of the following: daunorubicin 50 mg/m2 (DCE arm), mitoxantrone 12 mg/m2 (MICE arm), or idarubicin 10 mg/m2 (ICE arm). Patients in CR, received a single course of consolidation therapy, consisting in intermediate dose cytarabine (500 mg/m2) days 1 to 6, and the same intercalator used in induction, on days 4 to 6. After consolidation, those patients with an HLA-compatible sibling received an alloHCT while those without a sibling donor were randomly assigned to AuSCT. In the AML-12 trial, [2/12 h for four days) plus daunorubicin  and etoposide (100 mg/m2/d on day 1 to 5) or to DCE arm of AML-10 study. Patients obtaining CR received as the same consolidation regimen as AML-10 trial followed by alloHCT or AuSCT, depending on donor availability. From January 1998 to January 2008, patients were enrolled into the EORTC/GIMEMA AML-10 and AML-12 trials ,17. Pati2 as a daily continuous infusion for 10 days; i.v. etoposide 100 mg/m2/d on days 1 to 5 and, daunorubicin 50 mg/m2 on days 1, 3, and 5. Those in CR after 1 or 2 cycle of induction were given a consolidation therapy with cytosine arabinoside 500 mg/m2 twice a day, on days 1 to 6 and daunorubicin 50 mg/m2 on days 4 to 6. However, at variance with AML12 protocol, assignment to AuSCT or alloHCT was based on the integrated evaluation of baseline genetics/cytogenetics and immunophenotypic MRD status after consolidation cycle rather than on the donor availability. Since February 2008 to July 2013, due to conclusion of AML12 trial, patients were treated according to the standard-dose cytarabine AML12 arm. As an induction therapy, the patients received cytosine arabinoside 100 mg/mMore in detail, patients belonging to the intermediate genetics/cytogenetics were to receive AuSCT and alloHCT if MRD negative or positive, respectively. For those patients FLT3-ITD or bearing adverse karyotype an alloHCT had to be delivered. Patients allocated to AuSCT were to receive HDARAc in the case of stem cell mobilization or collection failure. On the other hand, patients qualified as high-risk were to receive an alloHCT whatever the source of stem cells . AuSCT or alloHCT were to be delivered within three months from the end of consolidation therapy. Patients enrolled in the GIMEMA AML1310 protocol  were notSince the retrospective nature of the study, informed consent for data collection was obtained from all subjects according to the specific per-protocol procedures of the trials involved in the present analysis.Therefore, for the purposes of the present analysis, we identified two patients’ cohorts: the MRD-driven cohort, in which post-consolidation assignment was risk-stratified as mentioned above, and the donor-driven cohort in which, per AML10/AML12 protocols, transplantation assignment relied only on the availability/lack of a familiar sibling donor, regardless of the individual risk profile . ®) given at dose of 5 mg/kg per day on days − 4 through − 1 in MUD and HRD. Graft-versus-host disease (GVHD) prophylaxis changed according to different sources of hematopoietic stem cells and included cyclosporine (CyA) plus i.v. methotrexate  in HLA-matched sibling and MUD. In HRD all patients received the same GVHD prophylaxis. It consisted of a combination of 5 drugs: anti-thymocyte globulin, CyA , methotrexate, mycophenolate mofetil (p.o. at 15 mg/kg/d from day 7 to 100). Furthermore, Basiliximab, an anti-CD25 monoclonal antibody, was given at fixed dose of 20 mg on days 0 and 4.High-dose busulfan combined with cyclophosphamide (BuCy) was the most commonly used conditioning regimen for AuSCT. As for as alloHCT, until the 2008, there were two main conditioning regimens, BuFlu and BuCy. From 2008, we adopted an identical conditioning regimen  for the MSD, MUD or HRD. The conditioning regimen included the anti-thymocyte globulin  samples were collected to perform immunophenotypic, chromosomal, and genetic studies as detailed elsewhere ,13,14,15criteria .2 test or Fisher’s exact test for categorical data. Relationship of MRD to treatment response was estimated by two-sided χ2 or Fisher’s exact test. OS was defined as the time interval from study entry until death from any cause or last follow up for patients alive. RFS was measured from the date of achievement of a remission until the date of relapse or death from any cause; patients not known to have relapsed or died at last follow-up were censored on the date they were last examined [p < 0.05 as indicating a statistically significant difference. All covariates were evaluated in univariate models and all factors with univariate association within p-value < 0.1 were considered in the multivariate models as potential parameters. Backward and stepwise methods were applied to identify the multivariate models with a step-by-step iterative construction that involves the selection of independent variables to be considered in the final model. Analyses were performed by SAS Version 9.2 statistical software  and R .Differences in the baseline characteristics were compared by χexamined . RFS andexamined . All tesp = 0.046). We also observed a higher frequency of NPM1 mutated patients in the MRD-driven cohort . As expected, more patients were addressed to alloHCT in the MRD-driven cohort than in the Donor-driven one . This was mainly due to the larger use of alternative sources of stem cells. In fact, sources of alloHCT were MSD in 27/49 (55%), MUD in 14/49 (29%) and HRD in 8/49 (16%) in the MRD-driven cohort whereas all four alloHCT in the Donor-driven cohort were MSD.Among 185 evaluable patients, 115 (62.2%) belonged to the MRD-driven cohort whereas 70 (37.8%) represented the donor-driven cohort. The clinical characteristics of the patients are detailed in p = 0.004) and NPM1-mutated . Accordingly, poor-risk karyotypes were more frequent in MRD positive (0% vs. 12.3%) and good-risk ones in MRD negative (40.5% vs. 9.2%) patients, respectively .MRD assessment was the additional biomarker considered for the risk stratification of the MRD-driven cohort. We further analyzed clinical and genetic prognosticators in this cohort according to MRD status . MRD posp = 0.012) and DFS  were longer in the MRD-driven cohort as compared to the donor-driven one (p < 0.001)  C.p = 0.0045), DFS  and CIR  were significantly better for the MRD-driven cohort than the Donor-driven one. In the analysis of the IR patients FLT3-ITD patients were not considered.When a subgroup analysis was performed, we observed a different impact of treatment strategy according to the karyotypic risk classes. Intermediate risk (IR) patients represented the category which benefited the most by the use of MRD-driven strategy . In factp = 0.0035) and DFS , with a lower CIR (p = 0.002). NRM was not statistically different between the two cohorts . The lack of statistically significant difference may well be explained by the higher non-relapse mortality (NRM) observed among the MRD-driven cohort , likely due to the higher number of alloHCT delivered (p = 0.042)  category. In fact, OS and DFS of FR patients A,B in thelivered C. Nevert= 0.042) D. With rDue to the role of MRD assessment in risk attribution, a separate analysis of the MRD impact on OS, DFS, CIR and NRM of every karyotype risk category was performed  improves outcome of adult patients with AML as compared to a Donor-driven approach.The critical role of prognostic biomarkers emerges especially when it comes to deciding as to whether a transplant should be delivered or not in first CR. In this view, the role of MRD has been a subject of discussion for a long time . Althoug−4 RLC after consolidation therapy had a worse outcome as compared to those MRD negative (<3.5 × 10−4 RLC). Submitting MRD positive patients to alloHCT, improved their prognosis in a way that the survival estimates equalized those of MRD negative ones submitted to chemotherapy and/or AuSCT [In our series, the superior outcome of IR patients belonging to the MRD-driven cohort is related to the high frequency of alloHCT delivery to patients who were MRD positive after consolidation cycle. IR category represents a clinical conundrum due to the genetic heterogeneity and the lack of definitive recommendations/guidelines about the transplant choice vs. alternative post remission approaches. In our former analysis, we observed that patients with a level of MRD ≥3.5 × 10or AuSCT ,15. Theror AuSCT . Altogetor AuSCT ,31,32. Ior AuSCT . The ideor AuSCT . This maBased on our experience, selecting the optimal post-remission regimen for FR patients (CBF-AML or NPM1 positive AML) poses several challenges. In the past, according to the “one size fits all” policy even patients with CBF-AML were submitted to alloHCT if a matched-sibling donor was available. With the time, it became evident that such an approach did not translate in an improved outcome since NRM counteracted the survival advantage determined by the procedure and the GVL . In a moA strategy of MRD-driven post-remission therapy determined an improved OS and DFS as compared to a non-risk-adapted strategy. Such an approach implies that alloHCT is delivered based on the actual risk of disease recurrence, according to a “transplant vs no transplant” rather than “donor versus no donor” policy. This, in turn, implies that once a given patient qualifies as high-risk, alloHCT is to be timely delivered whatever the source of stem cells. In FR patients, AuSCT may be a suboptimal choice if a status of MRD negativity is not reached.


To fight the novel coronavirus, one of the best-known ways is to block enzymes essential for virus replication. Currently, we know that the SARS-CoV-2 virus encodes about 29 proteins such as spike protein, 3C-like protease (3CLpro), RNA-dependent RNA polymerase (RdRp), Papain-like protease (PLpro), and nucleocapsid (N) protein. SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) for viral entry and transmembrane serine protease family member II (TMPRSS2) for spike protein priming. Thus in order to speed up the discovery of potential drugs, we develop DockCoV2, a drug database for SARS-CoV-2. DockCoV2 focuses on predicting the binding affinity of FDA-approved and Taiwan National Health Insurance (NHI) drugs with the seven proteins mentioned above. This database contains a total of 3,109 drugs. DockCoV2 is easy to use and search against, is well cross-linked to external databases, and provides the state-of-the-art prediction results in one site. Users can download their drug-protein docking data of interest and examine additional drug-related information on DockCoV2. Furthermore, DockCoV2 provides experimental information to help users understand which drugs have already been reported to be effective against MERS or SARS-CoV. DockCoV2 is available at  From December 2019 to June 2020, SARS-CoV-2 has infected over 10 million people, and caused >500 thousand deaths worldwide  includinThe SARS-CoV-2 genome contains ∼30 000 nucleotides that encode about 29 proteins such as spike protein, 3C-like protease , Papain-like protease (PLpro), RNA-dependent RNA polymerase , and nucleocapsid (N) protein . These fet al. used a computer-aided drug design and virtual screening to identify the mechanism-based inhibitor (N3) and the antineoplastic drug carmofur targeting Mpro  and the OpenData collected the actionable data and validated methodologies (https://doi.org/10.1101/2020.06.04.135046). Additionally, Covid19_DB lists the curated data of clinical trials in Covid-19/2019-nCoV (http://www.redo-project.org/covid19db/). To our knowledge, no database provides a more up-to-date and comprehensive resource with drug-target docking results for repurposed drugs against SARS-CoV-2.Drug repositioning is the process of finding new uses for existing approved drugs, and is believed to offer great benefits over  therapy . Here weFor drug repositioning, we selected FDA and Taiwan NHI  as our list of approved drugs. FDA-approved drugs were retrieved from the ZINC15 database , and Taihttps://doi.org/10.1101/2020.04.29.068890), RdRp (PDB ID: 7BV2)  , PLpro (D: 7BV2) , spike rD: 7BV2) , N proteD: 7BV2)  and ACE2D: 7BV2)  were retD: 7BV2) .DockCov2 aims to speed up the process of finding potential drugs by providing a computational representation of molecular docking with the most commonly-used drug databases . Throughet al. (https://amp.pharm.mssm.edu/covid19).The overview of the database is shown in Figure et al.  and COVIhttps://pubchem.ncbi.nlm.nih.gov/idexchange/). Specifically, multiple CIDs may be returned by PubChem Identifier Exchange Service for each drug name. We canonically chose the first returned record in order to be consistent with the result by manual search via the graphical interface of PubChem. Once we have the PubChem CIDs of our compound library, simplified molecular-input line-entry system (SMILES) (We chose PubChem compound identifiers (CIDs) as the key identifier in DockCoV2 because it is unique and widely used and can be easily linked to other external databases. For drugs retrieved from NHI, drug names were translated into CIDs using whole source mappings provided by UniChem  and PubC(SMILES) , interna(SMILES) .http://www.rdkit.org). Moreover, fragment screening is able to find a sensible starting point for the evolution of a new molecule in modern drug discovery. Previously, a large crystallographic fragment screen  were integrated into DockCoV2. PubChem CIDs were converted into DrugBank IDs first, then further converted into KEGG Drug IDs and KEGG Compound IDs. Alternatively, PubChem CIDs were directly converted to KEGG Drug IDs and KEGG Compound IDs by PubChem Identifier Exchange Service. Clinically related information including approval information, clinical phase, mechanism of action (MoA), target, disease area, and indication were also retrieved from the metadata file from the Drug Repurposing Hub.To provide useful validation information for drug repurposing and development, chemical information was aggregated either from external databases or directly calculated from structures. Three biological assays CHEMBL4303805, CHEMBL4303810, CHEMBL4303819 were fetched from ChEMBL . All of et al.  is a method that determines whether an a priori defined set of genes show statistically significant expression differences between two biological states . Here, tet al. . LiteratThere are many softwares that exist for virtual screening such as VSDK , AUDockehttp://autodock.scripps.edu/resources/adt) to convert the file format from MOL2 to PDBQT with default parameter settings excluding a parameter -A, which would add hydrogens to structure only if there are none already. After that, we applied rigid-body docking on these converted files using AutoDock Vina. In order to consider all of the potential docking poses, the entire protein is taken as the search space for blind search. We noted that the number of runs of the docking simulation should be adjusted accordingly by considering the variety of protein sizes. In AutoDock Vina, the number of runs is set by the exhaustiveness parameter, and the default setting of exhaustiveness is set at eight for a search space below 30 × 30 × 30 Å  format. The gen3d operation of OpenBabel (version 3.0.0)  was used0 × 30 Å . We prophttps://github.com/ailabstw/DockCoV2).After finishing docking, AutoDock Vina will generate multiple docking poses for each ligand–protein pair. For the sake of a straightforward representation of the docking results, the top 20 docking poses from AutoDock Vina were taken to build a protein heatmap. For each pose, a sphere centered at the centroid of ligand with a radius of 5 Å is highlighted, and the frequency of residues inside the sphere is incremented. The protein heatmap and the ligand docking position with the best affinity score are shown on the main page of each entry (a ligand–protein pair) in DockCoV2, where the red scale of the protein heatmap denotes the frequency of residues over twenty trials. Figure https://djangoproject.com) and Bootstrap 4.3.1 (https://getbootstrap.com/). The search bar, which can be found either in the index page or on navbar, allows the user to input the drug name, PubChem CID or synonym to explore the docking score with all proteins. The details of each ligand are shown after choosing a specific ligand–protein pair, including docking structure, ligand information and experimental data. PDBQT files of ligand and protein from AutoDock Vina are also provided on the main page of each ligand-protein pair. In the docking structure panel, both protein heatmap and sequences are displayed on the right-side of the detail page. Protein heatmap is interactively demonstrated with the NGL viewer tool, which is a WebGL-based molecular viewer  and molecular mechanics generalized Boltzman surface area (MM/GBSA) calculations . In addi